Kit for identifying bacteria from the Bacillus cereus group

ABSTRACT

The present invention relates to the field of microbiological testing of food. It relates to a kit comprising a reaction medium containing at least one inhibitor of Gram-negative bacteria and a fluorescent substrate specific for PC-PLC. It further relates to a diagnostic kit for identifying bacteria of the  Bacillus cereus  group comprising a container of a selective or nonselective reaction medium with a pH between 6.8 and 8.0, the medium comprising at least one inhibitor of Gram-negative bacteria and a fluorescent phosphatidylcholine phospholipase C (PC-PLC) substrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority as a divisional application of nationalstage application under 35 USC 371, Ser. No. 13/496,422, which was filedMar. 15, 2012, is now U.S. Pat. No. 8,871,465, and which claims thebenefit of the International Application No. PCT/ER2010/051925, filedSep. 16, 2010, which claims the benefit of French Patent Application No.0904470, filed Sep. 18, 2009, which are incorporated herein byreference.

Microbiological testing in the food-processing field requires theimplementation of techniques which allow the detection and/oridentification and/or counting of microorganisms, and the results ofwhich must be provided as rapidly as possible. Said microorganisms maybe nonpathogenic, such as bacteria of technological interest, forinstance ferments, or such as quality indicators; the latter make itpossible to validate the production process, from the raw materials orcrude products, to the final products. Nevertheless, said microorganismsare most commonly pathogenic, and the rapid and accurate detection ofpresumed contaminations makes it possible to take corrective steps.Alternatively, the toxins produced by said microorganisms andresponsible for pathogenic effects may be investigated.

Clinical diagnosis uses the same techniques: either the detection and/orcounting of the bacteria themselves, or the detection of the toxins. Inany event, the important factors for diagnostic tests are: sensitivity,selectivity and time for the result to be obtained.

The Bacillus genus comprises Gram-positive bacteria present ubiquitouslyin nature: in the ground, the water, the air, and also in food products,from cereal grains to powdered milks, floury products, spices. Theability to form spores gives them a very great resistance in theexternal environment. The spores of Bacillus cereus (B. cereus), inparticular, can soil foods, from the raw materials to the manufacturedproducts. Said spores survive throughout the length of the food chain.Under normal circumstances, B. cereus is present in an amount of lessthan 10³ cells per gram of food and has no pathogenic effect. Theminimum pathogenic level is greater than 10⁵ cells per gram of food. Thecontamination of an individual from a food can thus be responsible forgastroenteritis. Gastroenteritis associated with B. cereus is effectedeither by vomiting or by diarrhea. Various foods may be incriminated:meats, rice, dehydrated meals, sauces, soups, etc. Opportunisticinfections with B. cereus can also be observed in weakened patients,such as alcoholic or immunodepressed individuals or following injuriessuch as burns.

The detection and quantification of bacteria of the Bacillus cereusgroup is therefore essential for the testing laboratories of thefood-processing industry and for clinical diagnosis laboratories. In astandard manner, the isolation is carried out on conventional selectiveplating culture media: for example, standards of ISO (InternationalOrganization for Standardization) type or BAM-FDA (BacteriologicalAnalytical Manual of the Food and Drug Administration) methods recommendmedia such as polymyxin egg yolk mannitol bromothymol blue agar (PEMBA)or mannitol-egg yolk-polymyxin agar (MYP). The identification is carriedout according to morpholigcal characteristics and culture or metaboliccharacteristics.

These PEMBA or MYP media can lead to potential false positives linked toan inhibitor system that is not very effective, or to false negativeslinked to the possible absence of the key morphological and metaboliccharacteristics in certain strains. Finally, some strains exhibitambiguous reactions, as described by Fricker et al., InternationalJournal of Food Microbiology; 121 (2008): 27-34. Chromogenic platingmedia have been developed in order to overcome false negatives: theycontain chromogenic natural substrates or chromogenic syntheticsubstrates. Enzymatic activities specific for certain bacterial strainsare thus detected by cleavage of these substrates. The specificity ofthe detection can be improved by adding, to the culture medium,inhibiting systems, cocktails of antimicrobial and/or antifungal agents,intended to limit the growth of other microorganisms. However, theinhibitor cocktails also delay the growth of the target microorganisms,since they are intended to limit the growth of microorganisms of thesame genus as said target microorganisms.

Chromogenic or fluorescent media, based on the detection ofphosphatidylinositol-specific phospholipase C (hereinafter referred toas PI-PLC) activity, have been described in U.S. Pat. No. 6,284,517 B,EP 1 219 628 B and U.S. Pat. No. 6,558,917 B, and also in thepublication by Fricker et al., 2008 (above). These media have thedrawback of leading to false negatives, in particular with certainstrains of the Bacillus cereus group not exhibiting PI-PLC activity (B.cereus, B. mycoides, B. weihenstephanensis) or exhibiting weak PI-PLCactivity (B. anthracis), or to false positives. In addition, thefluorescent substrate 4MU-MIP (4-methylumbelliferylmyoinositol-1-phosphate) exhibits a reduced stability in an aqueousmedium which makes draconian conditions for use obligatory, inparticular a discontinuous measurement of the fluorescence, underprecise pH conditions, as indicated in U.S. Pat. No. 6,558,917. Moreprecisely, it is necessary to perform the culturing at acidic pH andthen to basify the medium in order to increase the fluorescence, whichis read at the end point. U.S. Pat. No. 7,309,580 B2 describes a platingmedium combining a chromogenic substrate for PC-PLC and a chromogenicsubstrate for PI-PLC. The respective colors, of the medium, of the firstsubstrate and of the second substrate are different and may also bedistinguished from the third color resulting from possible mixing of theenzymatic reaction products.

The BCM medium supplied by Biosynth® AG (Switzerland) uses a chromogenicsubstrate for PI-PLC and a system for inhibiting the non-targetedbacterial flora comprising polymyxin B, trimethoprim, sulfamethoxazoleand cycloheximide. The performance levels of the test are improvedcompared with the standard media, but some atypical strains can remainpoorly identified (Fricker et al., 2008, above).

Chromogenic media based on the hydrolysis of β-glucosidase substratesexist, such as Brilliance™ Bacillus cereus agar supplied by Oxoid™.However, said substrate generates Gram-positive false positives despitethe presence of an anti-Gram-positive inhibiting system comprisingpolymyxin B and trimethoprim, and also false negatives (Fricker et al.,2008).

Finally, a chromogenic plating medium, based on the hydrolysis of achromogenic substrate for PC-PLC, exists: R&F® Anthracis chromogenicagar. Said substrate provides a negative result indication for Bacillusanthracis in 24 hours and a potentially positive result in 48 hours. Thetime for obtaining the result remains long and the specificity requiresthe presence of several antibiotics.

It emerges from this review that, at the current time, there is nomethod for detecting and/or counting bacteria of the Bacillus cereusgroup using a reaction medium comprising an inhibitor of Gram-negativebacteria and a fluorescent substrate for PC-PLC, said reaction mediummaking it possible to obtain a result in 6 to 30 hours. Such a methodhas a real added value for clinical or industrial diagnosis, inparticular in the food-processing industry.

In view of the drawbacks noted in the prior art considered above, theessential objectives of the present invention are:

-   -   to obtain a positive result more rapidly than the existing        tests;    -   to reduce the number of false positives;    -   to improve the sensitivity, in particular for low levels of        contamination of the sample, through the use of a reduced        inhibiting system;    -   to provide very easy reading and interpretation through the use        of a single specific substrate, it being possible for said        reading to also be automated;    -   to provide simplified reaction and/or culture conditions, in        particular for the preparation of the medium and the stability        of the substrate.

According to a first embodiment, the invention relates to a method foridentifying bacteria of the Bacillus cereus group, comprising the stepsconsisting in:

-   -   bringing a sample that may contain bacteria of the Bacillus        cereus group, a reaction medium comprising at least one        fluorescent PC-PLC substrate and an inhibitor of Gram-negative        bacteria into contact, in a container;    -   incubating all the above together;    -   identifying the bacteria of the Bacillus cereus group by        detecting the PC-PLC substrate hydrolysis reaction,        in which the pH of the reaction medium and the time necessary        for detecting the PC-PLC substrate hydrolysis reaction carried        out are adapted in such a way that the said hydrolysis reaction        by the bacteria of the Bacillus cereus group is detected before        the hydrolysis of the PC-PLC substrate by the Gram-positive        bacteria other than those belonging to the Bacillus cereus        group, and potentially present in the sample, is detectable.

The expression “time necessary for detecting the substrate hydrolysis”is intended to mean the time which elapses between bringing the sample,the reaction medium and the substrate into contact in a container, anddetecting the signal. Concretely, this time is fixed and can be likenedto the reaction time. Said signal can therefore be considered to be aresult. The expression “before the hydrolysis of the PC-PLC substrate bythe Gram-positive bacteria other than those belonging to the Bacilluscereus group, and potentially present in the sample, is detectable” isintended to mean: before reaching, in the absence of bacteria of theBacillus cereus group, a signal corresponding to the threshold ofdetection of said bacteria of the Bacillus cereus group. The result isconsidered to be negative if the signal obtained is not significantlydifferent from the background noise; in other words, if it does notexceed the detection threshold. Said result is positive if the signalobtained is significantly different from the background noise.

Preferentially, the pH of the reaction medium is between 6.8 and 8.0.

Preferentially, the time necessary for detecting the PC-PLC substratehydrolysis is between 6 and 30 hours.

The reaction medium used in the method according to the invention ispreferentially in solid, liquid or gel form.

Advantageously, the reaction medium used in the method according to theinvention may be a culture medium.

Advantageously, counting of the bacteria of the Bacillus cereus groupthat are present in the sample is also possible.

According to one particular embodiment, the method according to theinvention can be carried out in microplates, microwells, microtubes,capillaries or multiwell cards such as the VITEK® or TEMPO® cardsdeveloped and marketed by the Applicant. Advantageously, the methodaccording to the invention may be combined with an automaticmicrobiological testing device of TEMPO® type as developed and marketedby the Applicant.

According to another embodiment, the method according to the inventionalso comprises a prior pre-enrichment step.

Advantageously, the reaction medium used in the method according to theinvention also comprises at least one second substrate, which ischromogenic or fluorescent. According to one particular embodiment, saidsubstrate is a PI-PLC substrate, which makes it possible to distinguishBacillus anthracis from Bacillus cereus and Bacillus thuringiensis.

Preferentially, the fluorescent PC-PLC substrate corresponds to 4 MU-CP(4-methylumbelliferyl choline phosphate).

According to one particular embodiment of the method according to theinvention, the bacteria of the Bacillus cereus group are chosen fromBacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillusmycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis, andthe other bacteria are chosen from Listeria monocytogenes, Listeriaivanovii, Staphylococcus or the other species of the genus Bacillusspp., such as Bacillus subtilis, Bacillus sphaericus, Bacilluscirculans, Bacillus lentus, Bacillus pumilus, Bacillus megaterium orPaenibacillus polymyxa.

The method which is the subject of the invention can be carried out bymeans of a kit comprising: a reaction medium containing at least oneinhibitor of Gram-negative bacteria and a fluorescent substrate specificfor PC-PLC. Said medium is resuspended with an aliquot of the sample tobe analyzed. Advantageously, the kit for carrying out the methodaccording to the invention can also contain a solid container ofmicroplate, microtube, microwell, capillary or multiwell card type, suchas VITEK® card or TEMPO® card, type. Preferentially, the fluorescentPC-PLC substrate used in the kit corresponds to 4-methylumbelliferylcholine phosphate (4 MU-CP). Advantageously, the kit for carrying outthe method according to the invention also comprises at least oneadditional substrate, which is chromogenic or fluorescent.Preferentially, said substrate is a PI-PLC substrate which makes itpossible to distinguish Bacillus anthracis from Bacillus cereus,Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus mycoidesand Bacillus pseudomycoides.

The specificity of a test is defined by the combination of thesensitivity and the selectivity. The sensitivity is defined as the powerto reveal the species being sought, when said species is present in asmall amount in a test sample. A low sensitivity will be reflected byfalse-negative results. The selectivity is defined as the power todetect the species being sought in the sample also containing otherspecies. The use of specific substrates for the metabolism of thespecies being sought improves the selectivity. However, strains whichexpress little enzymatic activity being sought may not be detected andmay lead to false-negative results. Similarly, the use of inhibitorcocktails, in other words of compounds capable of slowing down, limitingor blocking the growth of species potentially present, but the detectionof which is not desired, improves the selectivity. An inhibitor systemthat is not very effective can lead to false-positive results.

The term “sample” is intended to mean a small part or a small amountisolated from an entity for analysis. The sample may be of industrialorigin, that is to say, according to a nonexhaustive list, an airspecimen, a water specimen, a specimen taken from a surface, a part or amanufactured product, or a product of food origin. Among the samples offood origin, mention may, in a nonexhaustive manner, be made of a sampleof milk products (yogurts, cheeses, etc.), of meat, of fish, of eggs, offruit, of vegetables, of water or of a beverage (milk, fruit juice,soda, etc). These samples of food origin may also come from prepareddishes or sauces. Finally, a food sample may be derived from an animalfeed, such as in particular animal meals. The sample may be ofbiological origin, i.e. animal, vegetable or human origin. It may thencorrespond to a specimen taken from a biological fluid (whole blood,serum, plasma, urine, cerebrospinal fluid, organic secretion), a tissuespecimen or isolated cells. This specimen can be used as it is or, priorto analysis, undergo a preparation of enrichment, extraction,concentration or purification type, according to methods known to thoseskilled in the art.

Microbiological testing corresponds to the analysis of a sample with theaim of isolating and/or identifying and/or counting microorganismspotentially present, such as bacteria or yeasts. The term “reactionmedium” is intended to mean a medium comprising all the componentsnecessary for the survival and/or growth of the microorganisms. Thisreaction medium may either serve only as a revealing medium, or serve asa culture and revealing medium. In the first case, the microorganismscan be cultured before inoculation, and, in the second case, thereaction medium also constitutes the culture medium. The reaction mediummay be solid, semi-solid or liquid. The term “solid” is intended tomean, for example, a gelled medium. Preferentially, the medium accordingto the invention is a gelled medium. Agar is the conventional gellingagent in microbiology for culturing microorganisms, but it is possibleto use other gelling agents such as, for example, gelrite, gelatin oragarose. The reaction medium according to the invention may containoptional additives such as, for example: peptones, one or more growthfactors, carbohydrates, one or more selective agents, buffers, one ormore gelling agents, etc. This reaction medium may be in liquid form orgel form that is ready to use, i.e. ready for inoculation in a tube orflask or on a Petri dish.

Generally, the reaction medium may in addition contain a substrate fordetecting an enzymatic or metabolic activity of the targetmicroorganisms by means of a directly or indirectly detectable signal.For direct detection, this substrate can be linked to a part which actsas a label, which may be fluorescent or chromogenic. For indirectdetection, the reaction medium according to the invention may inaddition comprise a pH indicator, sensitive to the variation in pHinduced by the consumption of the substrate and revealing the growth ofthe target microorganisms. Said pH indicator may be a chromophore or afluorophore. As examples of chromophores, mention will be made ofneutral red, aniline blue and bromocresol blue. The fluorophorescomprise, for example, 4-methylumbelliferone, hydroxycoumarinderivatives or resorufin derivatives. Thus, the fluorescent PC-PLCsubstrate preferentially used for carrying out the method according tothe invention corresponds to 4-methylumbelliferyl choline phosphate (4MU-CP).

The method according to the invention will be better understood by meansof the examples below, which are in no way limiting in nature, incombination with the drawing in which:

FIG. 1 illustrates kinetic measurements of the PC-PLC activity ofvarious Gram-positive bacteria.

FIG. 2 illustrates the kinetic measurement of PC-PLC activity of theBacillus cereus ATCC 7064 strain as a function of the pH of the medium,respectively fixed at 7.2 or 6.8.

FIG. 3 illustrates the strength of coloration or of fluorescenceobtained in 24 h for various strains of Bacillus spp as a function ofthe type of substrate used.

EXAMPLE 1 Study of the PC-PLC Activity of Bacillus cereus Compared withthat Observed for Other Gram-Positive Bacteria (FIG. 1 and Table 1)

Various pure strains of the Bacillus genus and also other Gram-positivebacteria were tested in a microplate in the presence of the mediumbelow. A reading of the appearance of the fluorescence in each of thevarious wells of the microplate was then performed at different timesover the course of 44 h of incubation at 37° C.

1. Medium

The medium having the following composition was used (composition ing/l), pH 7.2:

Compounds Concentration in g/l Yeast extract 5 Sodium pyruvate 2Magnesium glycerophosphate 1 Basic HEPES buffer 13.8 Acidic HEPES buffer11.92 4-Methylumbelliferyl choline phosphate¹ 0.4 ¹4-Methylumbelliferylcholine phosphate (4 MU-PC), Biosynth ®, Ref. M-55282. Tests

The wells of the microplate are inoculated with 10 colony-forming units(CFU) of bacteria of the Bacillus cereus group and 1 000 CFU for theother Bacillus that are non cereus and other Gram-positive bacteria. Themicroplate is then incubated for 44 h at 37° C. in a microplate readerin order to evaluate the PC-PLC activity of these various strains in theform of kinetics of hydrolysis of the substrate 4 MU-CP, i.e to detectand measure the appearance of fluorescence. The detection threshold ofthe measuring apparatus is fixed at 30 000 RFU (relative fluorescenceunit).

3. Results and Interpretation

FIG. 1 shows a significant difference between the PC-PLC activityobserved in Bacillus cereus (positive signal from 15 hours onward) andthat observed in the other Gram-positive bacteria (positive signaldetectable only after 40 hours of incubation). It is not necessary toadd a complex inhibiting system to the medium in order to obtain thisresult.

These results were confirmed for 10 strains belonging to the Bacilluscereus group compared with 15 other strains not belonging to theBacillus cereus group. The latter results are collated in table 1.

It is therefore possible to distinguish the strains of the Bacilluscereus group compared with the other Gram-positive bacteria without acomplex inhibitor cocktail (presence only of an anti-Gram-negativeinhibitor) in a reading window of between 6 and 30 h.

Table 1: Level of fluorescence generated at 24 and 40 h by thehydrolysis of 4 MU-PC by various microorganisms. Detection threshold: 30000 RFU. The bacteria belonging to the Bacillus cereus group areindicated in bold characters.

Levels of Levels of fluorescence fluorescence Bacterial species in 24 h(RFU) in 40 h (RFU)

 ATCC 7064 >60000 >60000

 ATCC 6464 >60000 >60000

 ATCC 9139 >60000 >60000

 ATCC 10876 >60000 >60000

 ATCC 33019 >60000 >60000

 NCTC 11145 >60000 >60000

 0240015 >60000 >60000

 ATCC 6463 >60000 >60000 Bacillus licheniformis 93.08.043 2000 2000Bacillus sphaericus 8710054 20000 >60000 Bacillus circulans ATCC 451310000 20000 Bacillus subtilis ATCC 6051 10000 20000 Bacillus lentus ATCC10840 5000 10000 Bacillus pumilus ATCC 7061 5000 10000 PaeniBacilluspolymyxa ATCC 21551 5000 10000 Bacillus megaterium ATCC 14581 5000 5000L. monocytogenes ATCC 19118 4000 15000 L. monocytogenes 0301902 500010000 L. ivanovii ATCC 49954 2000 10000 L. ivanovii 0002147 2000 10000S. aureus 8407603 2000 2000 S. aureus 25923 2000 2000 Baseline 2000 2000

EXAMPLE 2 Study of the PC-PLC Activity of Bacillus cereus at pH 7.2Compared with pH 6.8 (FIG. 2)

The dynamics of the PC-PLC activity of Bacillus cereus ATCC 7064 wasevaluated at pH 7.2 (medium A) compared with pH 6.8 (medium B).

1. Media

Medium A has the following composition, pH 7.2:

Compounds Concentration in g/l Yeast extract 5 Sodium pyruvate 2Magnesium glycerophosphate 1 Basic HEPES buffer 13.8 Acidic HEPES buffer11.92 4-Methylumbelliferyl choline phosphate 0.4

Medium B has the following composition, pH 6.8:

Compounds Concentration in g/l Yeast extract 5 Sodium pyruvate 2Magnesium glycerophosphate 1 Na PIPES buffer 16.22 DiNa PIPES buffer17.32 4-Methylumbelliferyl choline phosphate 0.42. Tests

Ten CFU Bacillus cereus ATCC 7064 were inoculated into the wells of themicroplate in the presence of medium A (pH 7.2) and of medium B (pH6.8). The microplate is then incubated for 44 h at 37° C. in amicroplate reader in order to evaluate the PC-PLC activity of theBacillus cereus ATCC 7064 strain at the two pHs studied.

3. Results and Interpretation

The dynamics of the PC-PLC activity of Bacillus cereus ATCC 7064 and theRFU signals thus obtained at pH 7.2 are greater. Indeed, the 4 MUfluorescence emission strength increases with the pH of the medium(optimum pH for emission=10).

Taking into consideration the growth and the PC-PLC activity of Bacilluscereus, the optimum pH of the medium is 7.2. The obtaining of greatersignals therefore makes it possible to reduce the detection time, i.e by10 h in this specific case if the detection threshold is considered at30 000 RFU.

NB: in the case of the use of 4 MU-MIP (4-methylumbelliferylmyoinositol-1-phosphate, N-methylmorpholine salt, Biosynth®, Ref.M-5717) for distinguishing the bacteria of the Bacillus cereus group,this pH of 7.2 cannot be used owing to the instability of the substratebeing too great. A pH of 6.8 is therefore required, leading to anincrease in detection time.

EXAMPLE 3 Study of the Performance Levels of Various Substrates(Chromogenic Compared with Fluorogenic) for Revealing the PC-PLCActivity of Bacteria of the Bacillus cereus Group Compared with BacillusSpp. (FIG. 3)

Various strains of the Bacillus genus were tested on 10 different media.The plates are then read after 24 h and 48 h of incubation at 37° C.

The substrates tested are 5-bromo-4-chloro-3-indoxylcholine phosphate(X-CP), which is chromogenic, 5-bromo-6-chloro-3-indoxylcholinephosphate (magenta-CP), which is chromogenic, and 4-methylumbelliferonecholine phosphate (4 MU-CP), which is fluorescent.

1. Media

The medium having the following composition was used (composition ing/l):

-   -   yeast extract: 5 g/l    -   magnesium glycerophosphate: 1 g/l    -   agar: 13 g/l    -   LiCI: 3 g/l    -   MOPS: 12.6 g/l    -   MOPS sodium salt: 20.78 g/l

This medium was distributed into 10 different bottles (T, 1, . . . , 9)which were then sterilized by means of a 15 min/121° C. autoclave cycle.The medium T serves as a growth control. Stock solutions, at 30 g/l, of5-bromo-4-chloro-3-indoxylcholine phosphate (X-CP),5-bromo-6-chloro-3-indoxylcholine phosphate (magenta-CP) and4-methylumbelliferone choline phosphate (4 MU-CP) were prepared inosmosed water. Next, a volume corresponding to a final X-CPconcentration of 100, 300 and 900 mg/l, respectively, was added to themolten media denoted 1, 2 and 3, respectively. The same operation isrepeated for media 4, 5 and 6 and 7, 8 and 9 containing, respectively,100, 300 and 900 mg/l of magenta-CP and 100, 300 and 900 mg/l of 4MU-CP. These agar media were poured into Petri dishes.

2. Tests

The various Bacillus strains were inoculated by three-quadrant streakingusing suspensions at 0.5 McF (McFarland units). The dishes were thenincubated for 48 h at 37° C.

The colonies formed were examined visually after 24 and 48 h ofincubation. The coloration or fluorescence (read under a UV lamp at 366nm) of these colonies and also the strengths were noted.

3. Results

The coloration and fluorescence strengths are read on a relative scaleranging from 0 (no coloration/fluorescence) to 4 (very strongcoloration/fluorescence).

The results are illustrated in FIG. 3 (expression of the PC-PLC activityafter 24 h of incubation on various Bacillus species). The absence of abar indicates that the coloration or fluorescence strength measured isnot significantly different than the background noise, it is therefore anegative result.

4. Interpretation

The use of the fluorogenic PC-PLC substrate 4 MU-CP, unlike thechromogenic substrates (X-CP and magenta-CP) makes it possible to detectand distinguish bacteria of the Bacillus cereus group compared with theBacillus subtilis with high detection sensitivity and specificity(100%), in particular after 24 h of incubation at 37° C. and on all thestrains tested.

The invention claimed is:
 1. A diagnostic kit for identifying bacteriaof the Bacillus cereus group comprising a container of a selective ornonselective reaction medium with a pH between 6.8 and 8.0, said mediumcomprising at least one inhibitor of Gram-negative bacteria and a firstsubstrate, which first substrate is a fluorescent phosphatidylcholinephospholipase C (PC-PLC) substrate, wherein the reaction medium does notcontain an inhibitor of Gram-positive bacteria.
 2. The diagnostic kit asclaimed in claim 1, also comprising a container, selected from the groupconsisting of microplates, microwells, microtubes, capillaries andmultiwell cards.
 3. The diagnostic kit as claimed in claim 1, whereinthe fluorescent PC-PLC substrate corresponds to 4 MU-CP(4-methylumbelliferyl choline phosphate).
 4. The diagnostic kit asclaimed in claim 1, wherein the reaction medium also comprises at leastone second substrate, which is chromogenic or fluorescent.
 5. Thediagnostic kit as claimed in claim 4, in which the second substrate is aphosphatidylinositol-specific phospholipase C (PI-PLC) substrate whichmakes it possible to distinguish Bacillus anthracis from Bacilluscereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillusmycoides and Bacillus pseudomycoides.